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A Precisely Positioned MED12 Activation Helix Stimulates CDK8 Kinase Activity

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A Precisely Positioned MED12 Activation Helix Stimulates CDK8 Kinase Activity A precisely positioned MED12 activation helix stimulates CDK8 kinase activity Felix Klatta, Alexander Leitnerb, Iana V. Kima, Hung Ho-Xuanc, Elisabeth V. Schneiderd,e, Franziska Langhammera, Robin Weinmanna, Melanie R. Müllera, Robert Huberd,f,g,1, Gunter Meisterc, and Claus-D. Kuhna,1 aGene Regulation by Non-Coding RNA, Elite Network of Bavaria and University of Bayreuth, 95447 Bayreuth, Germany; bDepartment of Biology, Institute of Molecular Systems Biology, Eidgenössiche Technische Hochschule Zürich, 8093 Zürich, Switzerland; cBiochemistry Center Regensburg, Laboratory for RNA Biology, University of Regensburg, 93053 Regensburg, Germany; dMax Planck Institute of Biochemistry, 82152 Martinsried, Germany; eProteros Biostructures GmbH, 82152 Martinsried, Germany; fCenter of Medical Biotechnology, University of Duisburg-Essen, 45117 Essen, Germany; and gDepartment of Chemistry, Technical University of Munich, 85747 Garching, Germany Contributed by Robert Huber, December 21, 2019 (sent for review October 10, 2019; reviewed by Patrick Cramer and Matthias Geyer) The Mediator kinase module regulates eukaryotic transcription by with altered subunit composition accompanied by a potentially phosphorylating transcription-related targets and by modulating altered activity and target specificity (14). CDK8 and its paralog the association of Mediator and RNA polymerase II. The activity of its CDK19 were found to phosphorylate a wide variety of substrates catalytic core, cyclin-dependent kinase 8 (CDK8), is controlled by involved in transcription, DNA repair, and metabolic processes Cyclin C and regulatory subunit MED12, with its deregulation (11). This wealth of substrates likely lies at the heart of the context- contributing to numerous malignancies. Here, we combine in vitro dependent role of the Mediator kinase module in metazoan tran- biochemistry, cross-linking coupled to mass spectrometry, and scription. Moreover, the central role of CDK8 in transcription in vivo studies to describe the binding location of the N-terminal regulation explains why it is a potent oncogene that is involved in segment of MED12 on the CDK8/Cyclin C complex and to gain the progression of malignant cancers, like colorectal cancer and mechanistic insights into the activation of CDK8 by MED12. Our data acute myeloid leukemia (15–17). Due to its oncogenic potential, demonstrate that the N-terminal portion of MED12 wraps around the effective and selective inhibition of CDK8 is of great interest to CDK8, whereby it positions an “activation helix” close to the T-loop drug development and therefore spurred significant efforts to de- of CDK8 for its activation. Intriguingly, mutations in the activation velop CDK8-specific inhibitors. Despite the availability of structural helix that are frequently found in cancers do not diminish the affinity information on CDK8 bound to Cyclin C (18), the development of BIOCHEMISTRY of MED12 for CDK8, yet likely alter the exact positioning of the CDK8-specific inhibitors is hampered by the to date unclear activation helix. Furthermore, we find the transcriptome-wide mechanism of CDK8 activation. In contrast to the common acti- gene-expression changes in human cells that result from a mutation vation mechanism of cyclin-dependent kinases, CDK8 does not in the MED12 activation helix to correlate with deregulated genes in require phosphorylation of its T-loop for full activity. Instead, breast and colon cancer. Finally, functional assays in the presence of CDK8 T-loop phosphorylation seems to be functionally replaced by kinase inhibitors reveal that binding of MED12 remodels the active MED12 binding to CDK8, which results in its activation (5, 19). site of CDK8 and thereby precludes the inhibition of ternary CDK8 complexes by type II kinase inhibitors. Taken together, our results Significance not only allow us to propose a revised model of how CDK8 activity is regulated by MED12, but also offer a path forward in developing small molecules that target CDK8 in its MED12-bound form. CDK8, the catalytic core of the Mediator kinase module, is activated by MED12. We describe the architecture of the ternary complex of CDK8 | MED12 | Mediator kinase module | Mediator | kinase inhibitors CDK8, Cyclin C, and MED12, and we decipher the mechanism by which MED12 activates CDK8. We find the N-terminal segment of MED12towraparoundtheCDK8moleculetoplacean“activation ediator is essential for all RNA polymerase II (Pol II)- helix” in proximity of its T-loop. Exact placement of the helix, not Mmediated transcription in eukaryotes because it allows for MED12 binding alone, is crucial for CDK8 activation. Moreover, the communication between enhancer-bound transcription factors – MED12 binding precludes binding of type II kinase inhibitors to the and Pol II (1 4). In humans Mediator comprises 30 protein subunits active site of CDK8. As many cancer patients carry mutations in the that are subdivided into four modules: The head, the middle, the tail, MED12 activation helix, our study invigorates the development of and the kinase module. Whereas the first three modules form a CDK8-specific compounds as well as offers functional insights into complex that is often referred to as the Mediator complex, the kinase the Mediator kinase module. module only reversibly associates with the three-module Mediator – complex (5 7). Structural information on the isolated Mediator Author contributions: F.K. and C.-D.K. designed research; F.K., A.L., I.V.K., H.H.-X., E.V.S., complex and on the head and the middle module of Mediator, F.L., R.W., and M.R.M. performed research; G.M. and C.-D.K. supervised the study; F.K., collectively termed core Mediator, bound to Pol II provided first A.L., I.V.K., E.V.S., and R.H. analyzed data; and F.K., R.H., G.M., and C.-D.K. wrote insights into how Mediator is able to activate Pol II (8, 9). In contrast the paper. to the Mediator complex, the structure of the kinase module is un- Reviewers: P.C., Max Planck Institute for Biophysical Chemistry; and M.G., University known and its role during the transcription cycle is unclear. Despite of Bonn. the fact that, in yeast, the three-module Mediator complex is unable The authors declare no competing interest. to simultaneously bind Pol II and the kinase module (10), deletion or Published under the PNAS license. inhibitor of CDK8 results only in minor gene-expression changes (11, Data deposition: The mass spectrometry data were deposited to the ProteomeXchange 12). However, using human proteins in a mostly recombinant in vitro Consortium via the PRIDE partner repository (identifier PXD015394). The structure of Compound A in complex with CDK8 (1–403)/Cyclin C were deposited to the Protein Data system, the kinase module was found to repress transcription by Bank (PDB ID code 6T41). All sequencing data been deposited in the Gene Expression inhibiting Mediator coactivator function (13). Omnibus (GEO) database, https://www.ncbi.nlm.nih.gov/geo (accession no. GSE135458). The Mediator kinase module consists of four subunits: Cyclin- 1To whom correspondence may be addressed. Email: [email protected] or claus. dependent kinase 8 (CDK8), its corresponding cyclin, Cyclin C, and [email protected]. two large regulatory subunits, MED12 and MED13. Moreover, in This article contains supporting information online at https://www.pnas.org/lookup/suppl/ metazoans all kinase module subunits, except for Cyclin C, possess doi:10.1073/pnas.1917635117/-/DCSupplemental. paralogues that can be utilized for the assembly of kinase modules www.pnas.org/cgi/doi/10.1073/pnas.1917635117 PNAS Latest Articles | 1of12 Downloaded by guest on September 27, 2021 The current model for CDK8 activation proposes that the N-terminal necessary for CDK8 activation. In addition, we find that disease- segmentofMED12bindstoasurfacegrooveonCyclinC,andthereby causing MED12 mutations likely lead to mis-positioning of the enhances CDK8 activity (19). However, how MED12 binding to activation helix, and thereby abrogate CDK8 activity. In further this distant surface groove could be coupled to repositioning of support, we show that the transcriptome-wide gene expression the CDK8 T-loop remains unclear and the current model is changes in HCT116 cells that carry a MED12 E33Q mutation therefore controversial. phenocopy multiple cancer-related transcriptome alterations. Fi- To investigate how MED12 activates CDK8, we established a nally, we demonstrate that, in contrast to type I inhibitors, type II recombinant expression and purification scheme that resulted in kinase inhibitors lose much of their inhibitory potential when used monodisperse, highly pure ternary CDK8/Cyclin C/MED12 against ternary MED12-bound CDK8/Cyclin C in vitro. Taken complexes. Using these complexes, we carried out chemical cross- together, our results establish that an activation helix in MED12 linking coupled to mass spectrometry. Unexpectedly, the resulting functionally replaces T-loop phosphorylation of CDK8. Further- lysine- and aspartate/glutamate-mediated cross-links suggest a more, MED12 binding likely remodels the CDK8 active site, thus substantially revised mechanism for how the N-terminal portion of preventing type II kinase inhibitors from efficiently targeting MED12 binds and activates CDK8: MED12 makes extensive CDK8 bound by MED12 in vivo. contacts to CDK8 with no crucial contacts to Cyclin C. In- triguingly, MED12 thereby places a critical “activation helix” in Results direct vicinity of the T-loop of CDK8, leading to its conformational MED12 Binding Stabilizes
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